ABSTRACT Platelets play an essential role in the vessel, maintaining hemostasis and normal blood flow following vascular insult or injury under physiological conditions. While activation of the platelet is essential for adhesion and aggregation to occur at the site of vascular injury, excessive platelet reactivity can lead to the formation of occlusive thrombi, the predominant underlying cause of myocardial infarction and stroke. Current anti-platelet treatments have significantly limited morbidity and mortality due to thrombosis, however they often result in an increased risk of bleeding resulting in a need for novel targets to further decrease platelet reactivity while exhibiting a limited increased risk for bleeding. Our lab and others have provided compelling evidence supporting 12-lipoxygenase (12-LOX), an enzyme highly expressed in the platelet whose primary function is thought to be to produce bioactive oxidized lipids (oxylipins) from the fatty acids embedded in the platelet membrane, as playing an important role in the regulation of platelet activation. The role of newly studied fatty acids in the platelet such as DGLA, DHA, and EPA has yielded strong preliminary data supporting fatty acids and their 12-LOX oxylipins as being important for regulation of platelet function through GPCR and non-GPCR mechanisms. Our lab has identified the first 12-LOX negative regulation pathway in the platelet whereby formation of 12(S)-HETrE induces activation of a Gs-coupled GPCR pathway and activation of PKA resulting in inhibition of platelet activation and clot formation in vivo while not causing an observed increase in bleeding. We propose to investigate the underlying mechanisms by which these 12-LOX oxylipins regulate platelet activity, clotting, and thrombosis while only minimally affecting bleeding. Therefore, we will assess how DGLA and its oxylipin 12(S)-HETrE alters the phospho-proteome through activation of PKA, elucidate the mechanism by which DHA and EPA regulate platelet function and clotting both ex vivo and in vivo through their 12-LOX oxylipins while sparing hemostasis, and assess the mechanism by which the DHA 12- LOX oxylipin 14-HpDHA can be oxidized to form a new class of maresins, Mar1-D3, in order to regulate thrombolysis and clot resolution. Preliminary data suggests the platelet may produce a novel pool for this pro- resolving oxylipins that could play an important role in the underlying mechanism of clot resolution in vivo. Successful completion of this study will for the first time define the mechanism by which 12-LOX oxylipins regulate platelet reactivity in order to both prevent clot formation and resolve pre-existing clots. Understanding these complex signaling pathways represents a seminal advancement in our understanding of how oxylipins like 12(S)-HETrE regulate the blood and will enable for identification of new targets on the platelet for development of the next generation of anti-platelet therapeutics with the added benefit of limited bleeding.